[Alterations in the passive and active performance of cat papillary muscle induced by variation of preload (author's transl)]. In the isolated cat papillary muscle a rapid change in length induces a viscoelastic process of relaxation, during which the diastolic tension attains its new equilibrium after a delay. Its time course may be approximated both after stretching and releasing by a total of four exponential functions being marked by highly diverse time constants. During the stretch-induced relaxation phase the isometrically active papillary muscle shows a marked increase in mechanogram amplitudes, which is preceded in the first seconds by a short-term decrease. An opposite behavior is to be recorded after abrupt releases. The mechanograms of the stationary state prove to be exclusively a function of the degree of stretch, while the contractions in the early relaxation phases are dependent on the speed, direction and scale of the preceding change in length. The higher the stretching step chosen, the more clearly reduced are the mechanogram amplitudes of the early relaxation phase in comparison to the stationary state. This applies especially right of the optimum of force-development and contradicts a viscoelastic interpretation of the systolic phenomena in the poststretch phase. The findings after abrupt stretching point to either an initial decrease of amplitude or to a delayed approach of the contractions to their stationary state. Stretch-induced changes in the time course of the action potentials would constitute an adequate basis for the interpretation of these phenomena.


[Analysis of the dynamic action of vasoactive substances on the blood pressure of rabbits (author's transl)]. Rabbits were anesthetized with Urethane. The arterial blood pressure was recorded from the femoral artery with a Statham P 23 dB transducer. A servo-controlled Harvard Apparatus piston pump was used to infuse Acetylcholine and Noradrenaline respectively. The infusion rate was varied sinusoidally from 0.001 HZ to 0.1 HZ. The procedure was performed with different mean infusion rates and different infusion amplitudes. The response of the arterial blood pressure to the drug was examined by using the Bode analysis. The goal of our study was to examine if the blood pressure response to the infusion of a vasoactive drug can be characterized by a frequency dependent dose-response relationship. We could show that in terms of the frequency dependence the response to Noradrenaline differs in a quite typical way from the response to Acetylcholine. For both substances we were able to determine a frequency dependent transfer function. In addition we could describe the nonlinearity of the dose-dependent response of the blood pressure separately by an equation derived from the law of mass action. From the Bode diagrams the equations of the transfer functions are derived. In order to test the precision of the method, these equations were used to reconstruct the transfer function. This test is especially sensitive with respect to the phase of the function. We found a quite good agreement between calculated and measured values. This is a proof for the suitability of the method.


[A comparative study of the action of digoxigenin-mono, bis- and tridigitoxosides in the cat heart-lung preparation (author's transl)]. In experiments using spontaneously beating and electrically driven cat hearts (heart-lung preparations), the inotropic effects and therapeutic ranges of digoxin and its bis- and monodigitoxosides were compared. The myocardial contractility was measured by the maximal rate of left ventricular pressure rise, with regard to heart rate, enddiastolic left ventricular pressure, and diastolic aortic pressure. The therapeutic range was defined as the ratio of the arrhythmogenic concentration to the threshold concentration. On molar basis, an increasing cardioactivity in the order digoxin, digoxigenin-bisdigitoxoside, digoxigenin-monodigitoxoside was found. The monoditoxoside displayed the largest therapeutic range. The therapeutic range of bis-digitoxoside was similar to that of digoxin. The findings suggest that cardiac glycosides, their cardioactive derivatives and their cardioactive metabolites, respectively, do not possess identical therapeutic ranges.


[Data reduction, smoothing and base line correction preceding an automatic evaluation of ECG signals (author's transl)]. In storing electronically digital data of signals, too many items are used. Especially the great number of data produced in the analogue-digital-conversion of the ECG leads to serious problems in long distance transmission and storage. It is proposed to described such ECG signals through polynominal fragments, to which such a position across the noise-level distorted data is given that at the same time the data are levelled in a flexible manner. Based on these calculated reduced data, shape recognition problems can be solved more easily. As an example, a suggestion is given to construct the isoelectric segment of an ECG.


[Automated cytofluorometry of solid malignant tumors after cell dispersal by means of intraperitoneal cultivation of cell complexes in young rats (author's transl)]. The use of isolated cells is a basic requirement besides data processing techniques to obtain optimum results with the automated flow cytofluorometry. Several techniques have been employed in order to get cell dispersal. But the claim for reproducible and representative cell samples cannot exactly be fulfilled: gross parts of cells are damaged by any dispersal method applied on solid tissues. Therefore we want to introduce a special method for cell isolation, which avoids mechanical and chemical damage of the treated cells. For this the intraperitoneal tolerance of growing tumor cells in young animals was used, in the species of which solid growth of such tumor cells is established. The investigations reported here were carried out with the Walker-tumor 256 on the rat as a model for solid tumor growth. Our interest was focused on the determination of cellular CNA distribution in tumor cells and contaminating somatic cells grown in the ascitic fluids. Walker-tumors are permanently cultivated on Sprague-Dawley rats. The solid tumors (1 cm diameter) were removed under sterile conditions, cut with scissors, and resuspended in 0.9% saline. Ten rats (37 days old, body weight 90-120 g) had received an intraperitoneal injection of 1 ml Liquemin (5000 Roche). One million tumor cells could be obtained by such a procedure. After spontaneous sedimentation of cells in order to reduce the number of erythrocytes, the cells were processed and fixed according to Trujillo and Van Dilla (1972), digested with heated RNase (1 mg/ml tris-buffer; 0.18 M and pH 7.5), pepsin treated, and then stained with ethidium bromide (40 mug/ml tris-buffer) for 30 min in the cold. After rinsing with distilled water several times the cells were resuspended in buffer and measured within an hour immediately after passing a 10 mum filter to avoid cell aggregates...


[Effects of practolol on A-V conduction during atrial stimulation in 50 patients with and without coronary heart disease]. Practolol was injected intravenously at 3 dose levels (5, 15 and 25 mg total dose) in patients with ischemic heart disease and in non-cardiac cases. The influence of practolol on the P-Q interval at rest and at paced rates (atrial stimulation) of 110, 125 and 140 beats/min was studied: only the 15 mg dose produced a significant prolongation of the P-Q interval of normal patients at rest and at paced rates. No significant P-Q changes were found in patients with ischemic heart disease at all dose levels. It is concluded that practolol has no measurable direct effect on A-V conduction and that patients with ischemic heart disease are not more sensitive as regards A-V conduction.


Feulgen-DNA-values in transitional cell carcinoma of the human urinary bladder. Feulgen-stained imprint smears from 24 biopsy specimens from transitional cell carcinoma of the human urinary bladder were examined by means of scanning cytophotometry. One hundred randomly selected nuclei were measured from each biopsy specimen and the results compared with analogous measurements of nuclei from normal urinary transitional cell epithelium (13 cases in the control group). 97% of the nuclei in the control group were diploid. Well differentiated transitional cell carcinoma of the bladder mainly had a diploid DNA-stemline comprising 85% or more of the cells. One case of a well differentiated bladder cancer with a tetraploid DNA-stemline was found. The different cases of moderately differentiated bladder cancer showed diploid, triploid, tetraploid and hexaploid DNA-stemlines, while 4 out of 5 poorly differentiated tumours had a diploid DNA-stemline. The fifth extremely de-differentiated bladder carcinoma did not have any DNA-stemline at all. Together with these changes of the ploidy of the DNA-stemlines, increasing undifferentiation of the tumour tissue was combined with a reduced number of cells belonging to the tumour DNA-stemline, and increasingly scattered distribution of cells not contributing to the DNA-stemline. This indicates increased proliferative activity and/or chromosomal instability of the poorly differentiated cell population. Predominance of cells in the diploid, tetraploid and or octoploid intervals without marked frequency of DNA-values in the intermediary classes seemed to be a sign of clinical more "benign" bladder tumours, while a clinically more "malignant" tumour is characterized by increase of DNA-values in the triploid and hexaploid classes.


[Biomechanical investigations on the great veins (author's transl)]. Tensile breaking load, extensibility, elasticity and breaking energy of superior vena cava, inferior vena cava and portal vein of 33 men and 18 women have been determined in different directions by means of an electronic tension testing machine. 315 test specimens have been investigated with the following results: 1. The average breaking load in the tranverse direction of all three veins exceeds that in the longitudinal direction. 2. The maximum elongation is larger in the longitudinal than in the transverse direction. 3. The elastic constant is larger in the transverse than in the longitudinal direction, i.e. the extensibility is larger in the longitudinal direction. 4. Breaking load, elastic constant, and breaking energy are largest for the inferior vena cava and smallest for the portal vein. 5. The maximum elongation of the three veins is approximately equal. 6. No significant dependence of the obtained data on age was found.


Disturbances of karyokinesis and its relation to the nuclear pattern. The main causes of mitotic disturbances of spindle formation and inadaequate development of the mitotic apparatus. Quantitatively of the greatest importance in both groups are collapse of the prophase and failure of cytokinesis, respectively. The division of polyploid nuclei is, in addition, complicated by alterations of multicentric spindles. Results of mitotic disturbances are changes in the degree of ploidy of cells or nuclei. In irreversible post-mitotic tissues these occur already in the growth phase, as is shown in the example of the heart. In reversible post-mitotic tissues these changes in the degree of ploidy modify the nuclelar pattern predominantly during the processes of ageing, reparation and regeneration, as is well documented in the liver.


[The pulse-respiratory quotient of persons with stable and instable postural circulation while standing (author's transl)]. The pulse-respiratory quotient of patients with postural circulatory instability is the same in a prone position as that of patients with stable postural circulation, however, it is higher in an upright postiion. This is not only caused by the higher pulse frequency of postural circulatory instable patients while standing, but also by the lower respiratory rate. The difference of the pulse-respiratory quotient between standing and lying indicates with more accuracy the diagnosis of postural circulatory instability. The severity of the symptoms is related not only to the pulse and respiratory rate, but also to the pulse-respiratory quotient. Tachycardiac patients with a high pulse-respiratory quotient show clinically symptoms more severe than tachycardiac patients with lower pulse-respiratory quotients. Due to the results shown here, the terms of rhythmical concordance and discordance are proposed.


[Cytoplasmic tubuloreticular complexes and nuclear bodies in cells of rubella-infected human embryos and fetuses (author's transl)]. The range of single or multiple defects which might develop in the human embryo of fetus after an intra-uterine rubella infection has been documented by means of a large number of epidemiological, clinical and pathological investigations. Newborns with congenital rubella carry a number of defects, some of which could be the direct result of a virus-induced, intra-uterine vascular damage. In rubella-infected embryos and fetuses, vascular lesions are the most frequently observed histopathological finding. These changes have already been systematically investigated by means of the light microscope. Ultrastructural data are, however, not available at present.


Effect of cigarette smoking upon the finger circulation in normal and diabetic subjects. The basal finger blood flow and the reaction to cigarette smoking was studied among diabetic patients with neuropathy and non-diabetic controls. Both  the basal blood flow and the reaction to cigarette smoking were lower among diabetics. The diminution of the basal blood flow was believed to be secondary to the diabetic microangiopathy. The absence of vasoconstriction was attributed to a blockade of the neurologic mechanism involved. The site of the blockade remains open.


[Studies on the number of fat cells and connective tissue cells in white adipose tissue in chronic starvation and after refeeding. II. Investigations on obese and lean C 57 BL/6 J-mice (author's transl)]. Former investigations (Rakow et al., 1970, 1971a, b 1974) have demonstrated a constancy of the adipocyte number in white adipose tissue (parepididymal fat pads) of lean NMRI-albino-mice and aurothioglucose-obese NMRI-albino-mice during starvation and subsequent refeeding. In contrast the number of cells of connective tissue showed great variations under the experimental conditions mentioned above. The present paper describes which changes of the different cell populations within the adipose tissue could be demonstrated in lean and obese C57BL/6 J-mice. The investigations were performed with obese and lean male C57BL/6 J-mice. The control animal groups were fed for six weeks 2.5 g (lean mice) and 2 g (obese mice), respectively, Altromin 1115R daily (starvation phase). After this time some of these animalwere killed (exp. groups H). The remaining animals now were fed Altromin 1115R and additional oat falkes ad libitum. Three (exp. groups HW3) and seven (exp. groups HW 7) days, respectively, after the beginning of the refeeding phase the animals were killed. After sacrifice the epididymal fat pads were weighed and treated with either (fat extraction). The dry mass was hydrolized with PCA (0.5 m, 90 degrees C, 40 min). In the supernatant the DNA (Burton, 1956), RNA (Ceriotti, 1955) and polysaccharide content (Seifter et al., 1950) were measured. The sediment was hydrolized with NaOH (0.5 n, 37 degrees C, 24 hrs). In this solution the protein content (Lowry et al., 1951) was determined. In addition fat cells were isolated according to Rodbell (1964). The fat cell diameters were determined microscopically and the average masses of the fat cells were estimated. From the wet weight of the fat pads and the average fat cell mass and number of fat cells were calculated. The remaining suspension of fat cells and cells of connective tissue were utilized for cell smears. These cell smears were stained with Schiff's reagent (Feulgen et al., 1924; Graumann, 1953). With an integrating microdensitometer (Deeley, 1955) the average relative DNA-content of single cell nuclei was measured and the ploidy patterns were estimated. The DNA-content was measured chemically according to Burton (1956). From the whole DNA-content of the fat pads and the DNA-content of the fat cell population the number of cells of the connective tissue was calculated...


Ultrastructural studies on cells of hamster lung cultures after chronic exposure to whole smoke or the gas vapour phase of cigarettes. High levels of gas vapour phase constituents in cigarette smoke enhanced abnormal growth and tumorigenicity in primary cultures of hamster lung cells regularly exposed over long periods to the smoke from one of three types of cigarette. The ultrastructure of such cells was investigated by electron microscopy. Cells from cultures exposed to low levels of gas vapour phase constituents resembled cells from control, non-exposed cultures, and were filled with secondary lysosomes of the autophagic vacuole type. These autophagic vacuoles contained dense particles similar in size to the nucleoids of certain viruses. Cells from cultures exposed to high levels of gas vapour phase constituents and cells from tumours formed in nude mice after subcutaneous injection of transformed cultures lacked autophagic vacuoles but contained a higher number of cytoplasmic microfilaments. R-type (equals H-type) virus particles were found in the ER cisternae of control and exposure cultures at all ages, whilst A-type virus particles were found in close association with mitochondria in a transformed, exposed culture and in control cells which also formed tumours in nude mice but only after a much longer culture period.


Macroscopic ovarian involvement in murine plasmacytoma. In all ovaries of BALB/c/Se/Si mice carriers of plasmacytoma LPC/I, a plasmacellular proliferation was histologically observed. In 30% of the examined mice the proliferation had reached a macroscopical expression. This finding has not been previously observed.


The nature of inactivation of rat muscle 5'-adenylate aminohydrolase by fluorodinitrobenzene. 1. The inactivation of rat skeletal muscle AMP deaminase by Dnp-F (1-fluoro-2,4-dinitrobenzene) is accompanied by the arylation of thiol, amino and phenolic hydroxyl groups. 2. The number of thiol groups that react with Dnp-F is about 12; this is the number that reacts with Nbs2 [5,5'-dithiobis-(2-nitrobenzoic acid)] and N-ethylmaleimide without loss of enzyme activity, and it appears to be the same thiol groups that all three reagents attack. 3. Dinitrophenylation of these reactive SH groups is not the cause of inactivation, since active N-ethylmaleimide-substituted enzyme is also inactivated by Dnp-F.4. Complete inactivation of the N-ethylmaleimide-treated AMP deaminase occurs when about six tyrosine and two lysine residues are dinitrophenylated. 5. Since the treatment of Dnp-enzyme with 2-mercaptoethanol restores much of the enzyme activity, inactivation of AMP deaminase by Dnp-F is probably largely due to modification of tyrosine residues. 6. The kinetic properties of the Dnp-enzyme indicate that a marked decrease in V occurs only after extensive enzyme modification. The decreased activity after slight inactivation results from modification of Km.


Mechanistic, inhibitory and stereochemical studies on cytoplasmic and mitochondrial serine transhydorxymethylases. By using cytoplasmic and mitochondrial serine transhydroxymethylase isoenzymes from rabbit liver, it was shown that both enzymes exhibited similar ratios of serine transhydroxymethylase/threonine aldolase activities. Both enzymes catalysed the removal of the pro-S hydrogen atom of glycine, which was greatly enhanced by the presence of tetrahydrofolate. The cytoplasmic as well as the mitochondrial enzyme catalysed the synthesis of serine from glycine and [3H2]formaldehyde in the absence of tetrahydrofolate. The results are consistent with our previous suggestion that a role of tetrahydrofolate in the serine transhydroxymethylase reaction is to transport formaldehyde in and out of the active site (Jordan & Akhtar, 1970). The isoenzymes, however, showed remarkable differences in their inactivation by inhibitors. The serine transhydroxymethylase as well as the threonine aldolase activities of the cytoplasmic enzyme were inactivated in a similar fashion by chloroacetaldehyde, iodoacetamide, bromopyruvate and glycidaldehyde (2,3-epoxypropionaldehyde). These inhibitors had no effect on the two activities of the mitochondrial enzyme. The rate of inactivation of the cytoplasmic enzyme by glycidaldehyde was enhanced by the presence of glycine but decreased by the presence of serine. The implications of these results to the mechanism of catalysis and the nature of the active site of the enzymes are discussed.


Synthesis of cyclohexylpuromycin and its reaction with N-acetylphenylalanyl-transfer ribonucleic acid on rat liver ribosomes. 1. Cyclohexylpuromycin, an anlogue of puromycin in which a cyclohexane ring replaces the aromatic benzene ring of the L-phenylalanyl moeity of the nucleoside., has been synthesized and examined for its ability to release N-acetylphenylalanine from tRNA attached to rat liver ribosomes. 2.dl-Cyclohexylpuromycin was active in reacting with N-[3H]acetylphenylalanyl-tRNA on rat liver ribosomes to form N-E13H]lacetylphenylalanycyclohexypuromycin. 3. The reaction product N-acetylphenylalanylcyclohexylpuromycin and the corresponding analogue N-acetylphenylalanylpuromycin were chemically synthesized for evaluation of the structure of the released N-acetylphenylalanyl-containing material. 4. The results obtained suggest that the model of Raacke (1971) for purmycin reactivity needs further examination with regard to the role played by the aromatic ring system of the Lphenylalanyl moiety of the nucleoside


Separation by thin-layer chromatography and structure elucidation of bilirubin conjugates isolated from dog bile. 1. A system for separation of bile pigments by t.l.c. and for their structure elucidation is presented. Separated bile pigments are characterized by t.l.c. of derived dipyrrolic azopigments. 2. At the tetrapyrrolic stage hydrolysis in strongly alkaline medium followed by t.l.c. demonstrates the presence of bilirubin-IIIalpha, -IXalpha and -XIIIalpha and allows assessment of their relative amounts. 3. Most structural information is derived from analysis of dipyrrolic azopigments. Such derivatives, obtained by treatment of separated bile pigments with diazotized ethyl anthranilate, were separated and purified by t.l.c. Micro methods showed (a) the nature of the dipyrrolic aglycone, (b) the nature of the bonds connecting aglycone to a conjugating group, (c) the ratio of vinyl/isovinyl isomers present in the aglycone and, (d) the nature of the conjugating groups (by suitable derivative formation and t.l.c. with reference to known compounds). 4. In bile of normal dogs at least 20 tetrapyrrolic, diazo-positive bile pigments could be recognized. Except for two pigments the tetrapyrrolic nucleus corresponded predominantly to bilirubin-IXalpha. All conjugated pigments had their conjugating groups connected in ester linkage to the tetrapyrrolic aglycone, Apart from bilirubin-IXalpha, monoconjugates and homogeneous and mixed diconjugates of bilirubin were demonstrated; conjugating groups of major importance were xylose, glucose and glucuronic acid. 5. Bilirubin isomer determination on native bile and isolated bile pigments, and dipyrrole-exchange assays with [14C8]bilirubin indicated (a) that the conjugates pre-exist in bile, and (b) that no significant dipyrrole exchange occurs during isolation of the pigments.


Differences in the protein fluorecence of the two iron(III)-binding sites of ovotransferrin. 1. Changes in the tryptophan fluorescence and the visible absorption spectrum resulting from the combination of apo-ovotransferrin with Fe3+, F,E2+, Cu2+, Zn2+, Mn2+, and Cd2+were measured. 2. As expected for a radiationless transfer of electronic excitation energy, only the ions Fe3+, Fe2+and Cu2+, which gave complexes with large extinctions between 300 and 370nm, resulted in large decreases in trytophan fluorescence. 3. The decrease in protein fluorescence was non-linear with increasing occupancy of the Fe3+ -and Cu2+ - binding sites. The decrease in fluorescence on binding of Fe3+ was biphasic and showed that the two metal-binding sites were being occupied sequentially at pH7.4-8.4. The first site reacted with Fe3+ instantaneously, the second was occupied over a minute. 5. The nonidentity of the two sites was also demonstrated by the preparation of a stable hybrid containing both Cu2+ and Zn2+.h Cu2+ and Zn2+


The synthesis of hyaluronic acid by sheep and rabbit articular cartilage in vitro. Standard methods of glycosaminoglycan separation were used to confirm the presence of hyaluronic acid in sheep and rabbit articular cartilage. During incubation of carilage in vitro in the presence of [1-14 C]acetate, cartilage cells synthesize this macromolecule and, under the conditions used, it appears to have a shorter turnover time than chondroitin suplhate in the same tissue. The possible functions of hyaluronic acid in cartilage are discussed.


A comparsion of glycopeptides from the transferrins of several species. 1. The carbohydrate compositions of human, pig and cattle transferrins and duck ovotransferrin have been determined. 2. Glycopeptides have been prepared from these transferrins and their carbohydrate compositions and amino acid sequences determined. One of the glycopeptides from human transferrin carries the C-terminal residue of the protein. 3. Each tranferrrin yielded two glycopeptides that appeared to be identical in carbohydrate composition but different in amino acid sequence. The two glycopeptides have been distinguished as type A, in which the residue following Asn(CHO)(where CHO represents a carbohydrate moiety) is a basic amino acid and type B in which Asn(CHO) is followed by a neutral aliphatic amino acid. Cattle transferrin is exceptional in having two glycopeptides in which this position is occupied by serine. 4. It is suggested that each molecule of human and cattle transferrin and duck ovotransferrin carries an average of two carbohydrate prosthetic groups. Hen and pig transferrins appear to carry only one carbohydrate group per mol of protein. 5. The N-terminal sequences of hen and duck ovotransferrins and of cattle, human and pig transferrins were also determined.


Studies on the subunit structure of 4-deoxy-5-oxoglucarate hydro-lyase (decarboxylating) from Pseudomonas acidovorans. 1. Homogeneous preparations of D-4-deoxy-5-oxoglutarate hydro lyase (decarboxylating)(EC4.2.1.41) were analysed in the ultracentrifuge by the high-speed sedimentation-equilibrium method of Yphantis (1964). The molecular weight in 0.1 M-potassium phosphate buffer, pH 7.2, in 6M-guanidine hydrochloride and in 0.1 M-beta-mercaptoethanol in 6M-guanidine hydrochloride was 113,000, 56,000 and 30,400 respectively. Polyacrylamidegel electrophoresis in the presence of sodium dodecyl sulphate indicated a minimum molecular weight of 30,500. 2. Measurement of the thiol content of the enzyme, before and after reduction with NaBH4 or dithiothreitol under denaturing conditions, indicated the presence of eight thiol residues and two interchain disulphide bridges/enzyme molecule. 3. Amino acid analysis showed that the intact enzyme contains a total of approximately 100 arginine and lysine residues, but digestion of the enzyme with trypsin yielded about 49 peptides staining with ninhydrin  in a peptide "map". 4. With the knowledge that the enzyme contains only two substrate-binding sites, it is suggested that the enzyme probably consists of four polypeptide chains arranged in an alpha2beta2 confirmation.


Rat liver mitochondrial monoamine oxidase. A change in the reaction mechanism on solubilization. 1. The kinetics of benzylamine oxidation by a soluble preparation of rat liver mitochondrial monoamine oxidase were investigated and were shown to conform to adouble-displacement (or Ping Pong) mechanism. 2. The pathway differs in detail from that followed by other amine oxidases, including the membrane-bound enzyme in rat liver mitochondrial outer membranes. 3. It is suggested taht the conformation of the protein in the soluble state differs from that in the membrane-bound state. 4. The full rate equations for this mechanism have been deposited as Supplementary Publication SUP 50039 (5pages) at the British Library (lending Division) (formely the National Lending Library for Science and Technology), Boston Spa, Yorks, LS237BQ, U.K.. from whom copies can be obtained on the terms indicated in Biochem. J (1975) 145,5.


The role of a 5alpha-hydroxylated intermediate in the formation of the 5, 6-double bond in cholesterol biosynthesis. If the biological conversion of cholest-7-en-3beta-ol (I) into cholesterol (IV) occurred thorugh the intermediacy of cholest-7-ene-3beta,5alpha-diol (II) then the factor(s) adversely affecting the convwesion of the 5alpha-hydroxy sterol (II) into cholesterol must at least equally adversely affect the formation of cholesterol from cholest-7-en-3beta-ol. By using partial denaturation techniquws and dual-labelled precursors it was shown that the enzyme system responsible for the conversion of the 5alpha-hydroxy sterol (II) into cholesterol denatured faster than that for the corresponding conversion from cholest-7-en-3beta-ol (I).


The amino acid sequence of ribonuclease U2 from Ustilago sphaerogena. 1. RNAase (ribonuclease) U2, a purine-specific RNAase, was reduced, aminoethylated and hydrolysed with trypsin, chymotrypsin and thermolysin. On the basis of the analyses of the resulting peptides, the complete amino acid sequence of RNAase U2 was determined, 2. When the sequence was compared with the amino acid sequence of RNAase T1 (EC 3.1.4.8), the following regions were found to be similar in the two enzymes; Tyr-Pro-His-Gln-Tyr (38-42) in RNAase U2 and Tyr-Pro-His-Lys-Tyr (38-42) in RNAase T1, Glu-Phe-Pro-Leu-Val (61-65) in RNAase U2 and Glu-Trp-Pro-Ile-Leu (58-62) in RNAase T1, Asp-Arg-Val-Ile-Tyr-Gln (83-88) in RNAase U2 and Asp-Arg-Val-Phe-Asn (76-81) in RNAase T1 and Val-Thr-His-Thr-Gly-Ala (98-103) in RNAase U2 and Ile-Thr-His-Thr-Gly-Ala (90-95) in RNAase T1. All of the amino acid residues, histidine-40, glutamate-58, arginine-77 and histidine-92, which were found to play a crucial role in the biological activity of RNAase T1, were included in the regions cited here. 3. Detailed evidence for the amino acid sequence of the sequence of the proteins has been deposited as Supplementary Publication SUP 50041 (33 PAGES) AT THE British Library (Lending Division)(formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1975), 145, 5.


The subunit structure of beta-glucosidase from Botryodiplodia theobromae Pat. 1. A homologous series of beta-glcosidase (beta-D-glcoside glcohydrolase, EC 3.2.1.21), which varied in relative amounts in different preparations from cultures of similar and different age, was observed in cultures od Botryodiplodia theobromae Pat grown for 4-8 week on cotton flock (cellulose) as carbon source. 2. Aging of the purified high-molecular-weight species led to some amount of siddociation into a homolous series of lower-molecular-weight speices. 3. Rough molecular-weight estimates, by gel filtration, of the various species derived from the purifeid high-molecular-weight enzyme were 350000-3800000, 170000, 180000, 83000-87000 and 45000-47000. 4. Electron micrographs of the negatively stained 350000-380000-molecular-weight enzyme showed that the molecule is an octamer in which each roughly spherical monomer occupies a corner of a cube with each side about 7.14nm long. 5. Carboxamidomethylation of the reduced form of each molecular-weight species of the enzyme led to irreversible dissociation of the molecules into electrophoretically identical polypeptides with a moleclar weight of 10000-12000. 6. These results suggest a slow association-dissociation of the type (8n)in equilibrium 2 (4n) in equilibrium 4(2n) in equilibrium 8(n), where n is defined as the monomer. The monomer is in turn made up of four polypeptide a subunits whi-ch are non-catalytic. 7. The Michaelis constants (Km) and heat stability of the four wnzymically active molecular species derived from the purified enzyme increased with molecular complexity, whereas all four species were inhibited by glycerol (100nM)  at low concentrations of substrate (o-nitrophenyl beta-D-glucopyranoside) but activated at high substrat concentrations. 8. Only the lowest-molecular-weight species (45species (45,000-47000 mol. wt.) showed substrate inhibition.


The co-polymeric structure of pig skin dermatan sulphate. Distribution of L-iduronic acid sulphate residues in co-polymeric chains. 1. Pig skin dermatan sulphate was degraded by periodate oxidation followed by alkaline elimination or by chondroitinase-ABC to quantify irregular repeating units, i.e. those containing D-GlcUA (D-glucuronic acid) and L-IdUA-SO4 (sulphated iduronic acid). 2. Previous results of periodate oxidation (Fransson, 1974) indicated repeating sequences in pig skin dermatan sulphate containing, on average, 3D-GlcUA, 9 L-IdUA-SO4 or 28 L-IdUA units in addition to N-acetylgalactosamine sulphate. However, complete digestion with chondroitinase-ABC yielded, at the most, 3-4 disulphated disaccharides/chain. Consequently, more than one-half of the L-IdUA-SO4 residues were present in monosulphated periods, i.e. IdUA-(SO4)-GalNAc. 3. To determine the location of L-IdUA-SO4 residues along the copolymeric chain dermatan sulphate was digested with testicular hyaluronidase. (This enzyme cleaves GalNAc-GlcUA bonds within block regions containing D-GlcUA.) By NaB3H4 reduction GalNAc residues located in the reducing end of the fragments were converted into [3H]GalNAcOH (N-acetylgalactosaminitol). Finally, the radioactive product was fragmented by periodate oxidation followed by alkaline elimination. The bulk of the radioactivity was associated with periodate-resistant oligosaccharides indicating that clusters of GlcUA-GalNAc-SO4 periods are often adjacent to a varying number of (n = 1-4) of L-IdUA-SO4-containing periods. 4. To study the distribution of L-IdUA-SO4-containing periods in relation to blocks of IdUA-GalNAc-SO4 periods different fractions of hyaluronidase-degraded dermatan sulphate were degraded separately. In all types of fragments (mol. wts. 1,500-10,000) L-IdUA-SO4-containing periods were demonstrated. In short fragments reducing terminal GalNAc-6-SO4 (6-sulphated N-acetylgalactosamine) was found confirming that these sequences were joined to relatively long D-GlcUA-containing block sequences via GalNAc-6-SO4. Moreover, low-molecular-weight oligosaccharides composed of alternating sequences were encountered. An octasaccharide derived from the carbohydrate sequence -GalNAc---GlcUA-GalNAc-IdUA-GalNAc-GlcUA-GalNAc-IdUA-GalNAc---GlcUA-GalNAc (--- indicates the position of cleavage by hyaluronidase) was identified.


Hydrolysis of diepptide 2-naphthylamides by human muscle enzymes. Human muscle enzymes that hydrolyse dipeptide 2-naphthylamides were separated by chromatography on DEAE-Sephadex. Evidence was obtained that these substrates are hydrolysed by arylamidase as well as by dipeptidyl aminopeptidases.


Evidence for two asymmetric conformational states in the human erythrocyte sugar-transport system. 6-O-methyl-, 6-O-propyl-, 6-O-pentyl- and 6-O-benzyl-D-galactose, and 6-O-methyl-, 6-O-propyl- and 6-O-pentyl-D-glucose inhibit the glucose-transport system of the human erythrocyte when added to the external medium. Penetration of 6-O-methyl-D-galactose is inhibited by D-glucose, suggesting that it is transported by the glucose-transport system, but the longer-chain 6-O-alkyl-D-galactoses penetrate by a slower D-glucose-insensitive route at rates proportional to their olive oil/water partition coefficients. 6-O-n-Propyl-D-glucose and 6-O-n-propyl-D-galactose do not significantly inhibit L-sorbose entry or D-glucose exit when present only on the inside of the cells whereas propyl-beta-D-glucopyranoside, which also penetrates the membrane slowly by a glucose-insensitive route, only inhibits L-sorbose entry or D-glucose exit when present inside the cells, and not when on the outside. The 6-O-alkyl-D-galactoses, like the other nontransported C-4 and C-6 derivatives, maltose and 4,6-O-ethylidene-D-glucose, protect against fluorodinitrobenzene inactivation, whereas propyl beta-D-glucopyranoside stimulates the inactivation. Of the transported sugars tested, those modified at C-1, C-2 and C-3 enhance fluorodinitrobenzene inactivation, where those modified at C-4 and C-6 do not, but are inert or protect against inactivation. An asymmetric mechanism is proposed with two conformational states in which the sugar binds to the transport system so that C-4 and C-6 are in contact with the solvent on the outside and C-1 is in contact with the solvent on the inside of the cell. It is suggested that fluorodinitrobenzene reacts with the form of the transport system that binds sugars at the inner side of the membrane. An Appendix describes the theoretical basis of the experimental methods used for the determination of kinetic constants for non-permeating inhibitors.


Electrostatic effects on the kinetics of bound enzymes. 1. The effect of the interaction between the charged matrix and substrate on the kinetic behaviour of bound enzymes was investigated theoretically. 2. Simple expression is derived for the apparent Km. 3. The apparent Km can only be used for the characterization of the electrostatic effect of the ionic strength does not vary with the substrate concentration. 4. The deviations from Michaelis-Menton kinetics are graphically illustrated for cases when the ionic strength varies with the substrate concentration. 5. The inhibition of the bound enzyme by a charged inhibitor at constant ionic strength is characterized by an apparent Ki. 6. When both the inhibitor concentration and the ionic strength change there is no apparent Ki, and the inhibition profile is graphically illustrated for this case. 7. Under certain conditions the electrostatic effects manifest thenselves in a sigmoidal dependence of the enzyme activity on the concentration of the substrate or inhibitor.


Sensitive analysis of ethanolamine- and serine-containing phosphoglycerides by high-performance liquid chromatography. A highly sensitive method for the separation and quantitative measurement of phospholipids containing primary amino groups, such as phosphatidylethanolamine, phosphatidylserine and lysophosphatidylethanolamine, is described. The method involves a simple and quantitative derivative formation of the phospholipids containing amino groups to their u.v.-absorbing biphenylcarbonyl derivatives. These have molar extinction coefficients of about 23,000 at 268nm. The phospholipid derivatives are then separated and non-destructively determined by high-performance liquid chromatography. The amino phospholipids containing vinyl ether bonds (plasmalogens) can be determined separately from the diacyl- and alkylacyl-amino phospholipids. The lower limit of detection by high-performance liquid-chromatographic analysis of the phospholipid derivatives is about 10-13pmol or 0.3-0.4ng of phospholipid P. The quantitative range of derivative formation and analysis by high-performance liquid chromatography of the phospholipids containing amino groups was shown to be 10-500nmol. The method was shown to be applicable to the analysis of phospholipids containing amino groups in tissue samples.


Microtubule protein synthesis during oogenesis and early embryogenesis in Xenopus laevis. A method is described which permits the preparation of descrete classes of oocytes of different sizes from all stages of oogenesis in Xenopus laevis. This technique is used in the determination of the content of microtubule protein in oocytes during the course of oogenesis. These experiments show that microtubule protein is present in oocytes of all sizes assayed and that the amount is simply related to the volume of the oocyte. In the largest oocytes microtubule protein constitutes 1% of the soluble protein and this amount does not change on maturation and fertilization. These results show that the changes occurring in the oocyte on maturation which allow the cytoplasm to support microtubule polymerization occur as a result of a modification of the pre-existing microtubule protein, not from protein synthesis de novo. These experiments also indicate that the synthesis of microtubule protein either form 'masked' mRNA or from newly synthesized mRNA plays an insignificant role in microtubule protein synthesis at maturation, ovulation and immediately post-fertilization.


Chemical and physical characteristics of a phosphoprotein from human parotid saliva. The isolation of a highly purified phosphoprotein, previously named protein A, from human parotid saliva is described. This protein has an unusually high amount of glycine, proline and dicarboxylic amino acids. Together these amino acids account for 80% of all residues. The protein contains 1.9mol of P/mol of protein, probably as phosphate in an ester linkage to serine, and about 0.5% carbohydrate, but no hexosamine. The N-terminal is blocked and the following C-terminal sequence is proposed: -Aal-Asp-Ser-Gln-Gly-Arg-Arg. The sioelectric point is 4.43. The molecular weight of the protein determined by ultracentrifugation is 9900 and from chemical analyses 11000. Circular-dichrosim and nuclea-magnetic-resonance spectra indicate the absence of polyproline and triple-helical-collagen-like structure for the protein. There is little restriction on the orientation of the single phenylalanine residue in the protein., but there is also an indication of conformational restraint in the protein.


Comparison of the subunit and primary structures of the pyruvate kinases from rabbit and sturgeon muscles. The structures of the pyruvate kinases isolated from rabbit and sturgeon muscles were compared. Both enzymes are composed of subunits of 56000 mol.wt. Amino acid compositions of the two enzymes are similar, but not identical. Examination of the peptides produced by CNBr cleavage demonstrated that there are at least some highly homologous regions in the two proteins. There are only two replacements between an 18-residue portion of the polypeptide chain of rabbit muscle pyruvate kinase and a portion of the polypeptide chain of the enzyme isolated from sturgeon muscle.


Hybridization properties of nucleolar ribonucleic acid. Rat liver nuclei were fractionated into chromatin and nucleolar fractions. Chromatin DNA, which does not form hybrids with rRNA, was, nevertheless, able to hybridize with 32P-labelled total nucleolar RNA. The optimal temperature for this hybridization was 55 degrees C when the reaction was carried out in 2 X SSC (0.3 MnaCl + 0.3 M-sodium citrate). The hybrids formed were specific, as judged by analysis of thermal elution profiles. The low Tm (73 degreesC) observed could be explained by the low amount of DNA in the filters. The lenth of the hybridized sequences was extimated as 54 mucleotide pairs. Contamination to nucleolar RNA by nucleoplasmic RNA was ruled out by showing the former was able to form more hybrids than the latter. Competition experiments showed that hybridization of nucleolar RNA, although not competed with by rRNA, suffered pronounced competition from total microsomal RNA, even though the levels of competition obtained did not equal thsoe with cold nucleolar RNA as competitor.


Glycosylation of ribonuclease A catalysed by rabbit liver extracts. Crude extracts of rabbit liver catalyse in vitro the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to bovine pancreatic ribonuclease A. The enzymic activity is contained in rough endoplasmic reticulum. It has an absolute requirement for a bivalent metal ion: Co-2+ greater than Mn-2+ greater than Ni-2+. Mg-2+ is ineffective. There is enzymic activity in the absence of detergent, but increased activity is observed in the presence of Triton X-100. The site of glycosylation of ribonuclease A is asparagine-34, and glycosylation occurs only at this point. These findings agree with the hypothesis that the sequence Asn-X-Thr(Ser) (where X may be one of a number of types of amino acid) is a necessary, but not sufficient, condition for N-acetylglucosaminylation of a protein-bound asparagine residue.


Increased synthesis of low-molecular-weight nuclear ribonucleic acids of rat liver after gamma irradiation and hepatectomy. Incorporation of [3H]orotic acid into low-molecular-weight nRNA of rat liver, fractionated on polyacrylamide gels, increased 6-12h after partial hepatectomy and 6h after gamma-irridation at 2000 R. The incorporation of orotic acid was particularly increased into the 4.5S, 5S and approx. 10S nRNA fractions. If the irradiation was given after 6h of regeneration and RNA was isolated from the nucleus 12h after hepatectomy then the incorporation of orotic acid into these low-molecular-weight nRNA components was greater than after hepatectomy or irradiation alone.


Electrophoretic and centrifugation behaviour of mitochondrial ribonucleic acid from Walker 256 carcinosarcoma. To investigate the possibility that mitochondrial transcription could be altered in tumours we started by characterizing the RNA obtained from mitochondria, isolated from Walker carcinosarcoma and purified by a procedure devised to compensate for the lower size and density of these organelles in 10-day tumours. The RNA was extracted by the 'hot phenol' technique and analysed by electrophoresis in 2.7 and 2.5% polyacrylamide gels at different running times, identifying the usual cytoplasmic contaminants 28 and 18S peaks plus the other five major peaks at 40, 20.5, 16.3, 15.4, and 4Se. The 28 and 18Se peaks were not eliminated by digitonin treatment of the mitochondria, indicating that they arise from cytoplasmic ribosomes tightly associated with the mitochondria. From its sensitivity to DNAase (deoxyribonuclease), resistance to RNAase (ribonuclease) and coincidence with external marker DNA, the 40Se peak was identified as containing mainly DNA. Sucrosegradient centrifugation for different periods showed a major component at 16.2S, the 28 and 18S cytoplasmic RNA species, peaks at 13.8, 6.4 and 4S and a small 19.5S peak. By polyacrylamide-gel electrophoresis of the purified RNA classes separated by one or two cycles of centrifugation, the following correlation were established: 20.5Se19.5S; 16.3Se16.2S; 15.4Se13.8S. The 6.4S RNA ran as a mixture of 4 and 4.7Se species. When the 20.5Se and 15.4Se RNA species were centrifuged, they behaved as 16.2S and 13.8S respectively, thus suggesting that the 16.2S (16.3Se) arises by cleavage from the 19.5S(20.5Se), the 13.8S (15.4Se) being the other RNA from mitochondrial ribosomes.


Biosynthesis of serum albumin in rat liver. Isolation and probable structure of 'proalbumin' from rat liver. 1. Two methods are described for the preparation of 'proalbumin' in good yields from rat liver. 2. One of the methods does not depend on the use of specific antisera. 3. The product from both methods is identical as judged by electrophoresis on polyacrylamide gel, isoelectric focusing on pH gradients, ion-exchange chromatography and quantitative immunoelectrophoresis. The protein also appears to be radiochemically pure by these criteria. 4. The protein is free from serum albumin as judged by isoelectric focusing and co-chromatography on ion-exchange columns. It is judged to be free from other proteins by these same criteria and by specific precipitation with antibody. 5. It is converted into serum albumin by limited tryptic hydrolysis. The albumin so produced has the same N-terminal (glutamic acid) and C-terminal (alanine) amino acids as reported for rat serum albumin. 6. A hexapeptide is liberated from the N-terminal end of 'proalbumin' simultaneously. It contains three arginine, one phenylalanine, one valine and one glycine residues.


Transfer of serine into polypeptides and myosin by chromatographic species of seryl-transfer ribonucleic acid. The efficiencies of two chromatographic species of [3-H]seryl-tRNA, namely peaks I and II, in cell-free amino acid incorporation were investigated. The maximum yield of polypeptide seems to be the same for the reaction mixtures containing either peak I or peak II, suggesting that the efficiency of both peaks in total protein synthesis is the same. The efficiency of transfer of serine into myosin heavy subunit (myosin H) by peaks I and II was also investigated. Peak II of [3-H]seryl-tRNA transfers three times as much serine into myosin H as peak I.


The synthesis of fatty acids in avocado mesocarp and cauliflower bud tissue. 1. Plastid and mitochondrial preparations were obtained by density-gradient centrifugation of homogenates made by gentle disintergration of avocado fruit mesocarp and cauliflower bud tissue. 2. The mitochondrial preparations had respiratory activity but did not incorporate [1-14C]acetate into fatty acids. 3. The plastid preparations incorporated [1--14C]acetate into the range of fatty acids found in the parent tissue. No fatty acid synthetase activity could be detected in the 12000g supernatant of these homogenates. 4. Homogenates produced by rupture of the tissue in an Ato-Mix blender and plastid preparations disintegrated by ultrasonic treatment both had fatty acid synthetase activity which did not sediment at 105000g and which formed mainly [14-C]stearate from [2-14C]malonyl-CoA. 5. It is concluded that the plastids are the principal site of fatty acid biosynthesis in the tissues studied.


The fractionation of the fatty acid synthetase activities of avocado mesocarp plastids. 1. The range of fatty acids formed by preparations of ultrasonically ruptured avocado mesocarp plastids was dependent on the substrate. Whereas [1-14C]palmitate and [14C]oleate were the major products obtained from [-14C]acetate and [1-14C]acetyl-CoA, the principal product from [2-14C]malonyl-CoA was [14-C]stearate. 2. Ultracentrifugation of the ruptured plastids at 105000g gave a supernatant that formed mainly stearate from [2-14C]malonyl-CoA and to a lesser extent from [1-14C]acetate. The incorporation of [1-14C]acetate into stearate by this fraction was inhibited by avidin. 3. The 105000g precipitate of the disrupted plastids incorporated [1-14C]acetate into a mixture of fatty acids that contained largely [14C]plamitate and [14C]oleate. The formation of [14C]palmitate and [14C]oleate by disrupted plastids was unaffected by avidin. 4. The soluble fatty acid synthetase was precipitated from the 105000g supernatant in the 35-65%-saturated-(NH4)2SO4 fraction and showed an absolute requirement for acyl-carrier protein. 5. Both fractions synthesized fatty acids de novo.


Thymidine kinase of mouse spleen cells in vivo and in vitro. The thymidine kinase activity of mouse spleen cells was found to parallel their DNA-synthesizing ability, both in vivo and in vitro. In the former case, more than 90% of the activity of these cells was found in the cytoplasmic fraction. This activity was labile, as was its template mRNA, compared with the nuclear component. The nuclear enzyme increased during culture of the lymphocytes in vitro. Mitogenic stimulation with concanavalin A resulted not only in a greater enhancement of the nuclear activity, but also in a marked increase in the amount of cytoplasmic enzyme. This effect appeared to be mediated via stabilization of the mRNA for the cytoplasmic component. These differences have been considered, especially with respect to the cellular changes that occurred during culture and mitogenic stimulation.


The occurrence of a phosphorylated glycosphingolipid in Aspergillus niger. A novel type of water-soluble phosphorylated glycosphingolipid was isolated from Aspergillus niger by a simple procedure involving precipitation, DEAE-cellulose chromatography and preparative t.l.c. Besides ceramide and phosphorus it contains inositol, galactose, mannose and small amounts of glucosamine.


Effect of allylisopropylacetamide on Nuclear Ribonucleic Acid synthesis in rat liver. The porphyrogenic drug allylisopropylacetamide, a potent inducer of delta-aminolaevulinate synthetase, specifically increases nucleoplasmic RNA synthesis in rat liver. The drug-mediated increase in nucleoplasmic RNA synthesis is blocked by cycloheximide and haemin, which also inhibit the enzyme induction.


The infrastructure of valyl-transfer Ribonucleic Acid synthetase from yeast. Valyl-tRNA synthetase from yeast is shown to contain a significant proportion of repeated sequence, similar to the enzyme from bacterial sources.


The effect of zinc on the activity and fluorescence of carbonic anhydrase holoenzymes. The addition of Zn2+ to human carbonic anhydrase B holoenzyme was shown to enhance the protein fluorescence, and this enhancement was correlated with the inhibition of the p-nitrophenyl acetate esterase activity. The affinity for the inhibitory Zn2+ was increased when the ionic inhibitors, acetate or chloride, were added, suggesting that the inhibitory Zn2+-binding site is within the region of the protein that undergoes an anion-induced conformational change. A similar fluorescence enhancement was observed when Zn2+ was added to human carbonic anhydrase C and to bovine carbonic anhydrase, demonstrating that the binding site is not a thiol group. Circular-dichroism studies showed that the C isoenzyme but not the B isoenzyme underwent a major conformational change in the presence of Zn2+. A mechanism for the Zn2+-induced fluorescence enhancement was suggested on the basis of studies with simple compounds.


Characterization of jack-bean alpha-D-mannosidase as a zinc metalloenzyme. 1. Two methods were used to obtain alpha-mannosidase free from unbound Zn2+, (a) by removal of excess of metal ion from preparations purified in the presence of Zn2+ and (b) by purification under conditions that eliminate the need to add Zn2+. 2. The purified enzyme is homogeneous on ultracentrifugation, polyacrylamide-gel electrophoresis and gel chromatography. 3. The molecular weight is estimated to be 230 000. 4. The enzyme contains between 470 and 565 mug of zinc/g of protein, corresponding to between 1.7 and 2 atoms of zinc/enzyme molecule. The contents of other metals are much lower. 5. The enzyme is inactivated by chelating agents and activity is restored by Zn2+. 6. No other metal ion was found to replace Zn2+ with retention of activity. Some bivalent metal ions, e.g. Cu2+, rapidly inactivate the enzyme. 7. The results indicate that jack-bean alpha-mannosidase exists naturally as a zinc-protein complex and may be considered as a metalloenzyme.


The effects of oestradiol-17beta on the ribonucleic acid polymerases of immature rabbit uterus. Measurements of the endogenous RNA polymerase activities of nuclei isolated from immature rabbit uteri have shown that prior treatment of the animals with oestradiol-17beta has a profound effect on the apparent activities of both RNA polymerases A and B. Within 1 h of hormone treatment, the activity of RNA polymerase A is increased and continues to rise until about 4h when it reaches a plateau and remains steady until at least 8h. The activity of RNA polymerase B increases sharply after oestradiol treatment reaching an early maximum at 30-45 min. Thereafter this activity declines until by 1-2h it approaches control values but a second increase in activity then occurs with a maximum at 3-4h. Treatment of the rabbits with alpha-amanitin before the administration of oestradiol inhibits the hormone-induced stimulation of RNA polymerase A activity in isolated nuclei but when the administration of alpha-amanitin is delayed until after the early rise of RNA polymerase B activity, the oestradiol-induced stimulation of RNA polymerase A is retained. Similar results have been obtained in experiments with cycloheximide suggesting that the stimulation of RNA polymerase A activity by oestradiol is dependent on the hormone-induced stimulation of RNA polymerase B and the subsequent synthesis of protein using the RNA product of the early increase in RNA polymerase B activity. Measurement of the activities of RNA polymerases A and B after isolation of the enzymes from immature rabbit uterine nuclei before and after oestradiol treatment failed to show any differences. Therefore it would appear that the changes in the observed activities of RNA polymerases A and B in isolated nuclei are consequences of changes in the structure and function of chromatin rather than the results of modifications in the RNA polymerases themselves.


The metabolism of benzoate by Moraxella species through anaerobic nitrate respiration. Evidence for a reductive pathway. Moraxella sp. isolated from soil grows anaerobically on benzoate by nitrate respiration; nitrate or nitrite are obligatory electron acceptors, being reduced to molecular N2 during the catabolism of the substrate. This bacterium also grows aerobically on benzoate. 2. Aerobically, benzoate is metabolized by ortho cleavage of catechol followed by the beta-oxoadipate pathway. 3. Cells of Moraxella grown anaerobically on benzoate are devoid of ortho and meta cleavage enzymes; cyclohexanecarboxylate and 2-hydroxycyclohexanecarboxylate were detected in the anaerobic culture fluid. 4. [ring-U-14C]Benzoate, incubated anaerobically with cells in nitrate-phosphate buffer, gave rise to labelled 2-hydroxycyclohexanecarboxylate and adipate. When [carboxy-14C]benzoate was used, 2-hydroxycyclohexanecarboxylate was radioactive but the adipate was not labelled. A decarboxylation reaction intervenes at some stage between these two metabolites. 5. The anaerobic metabolism of benzoate by Moraxella sp. through nitrate respiration takes place by the reductive pathway (Dutton & Evans, 1969). Hydrogenation of the aromatic ring probably occurs via cyclohexa-2,5-dienecarboxylate and cyclohex-1-enecarboxylate to give cyclohexanecarboxylate. The biochemistry of this reductive process remains unclear. 6. CoA thiol esterification of cyclohexanecarboxylate followed by beta-oxidation via the unsaturated and hydroxy esters, would afford 2-oxocyclohexanecarboxylate. Subsequent events in the Moraxella culture differ from those occurring with Rhodopseudomonas palustris; decarboxylation precedes hydrolytic cleavage of the alicyclic ring to produce adipate in the former, whereas in the latter the keto ester undergoes direct hydrolytic fission to pimelate.


The aerobic metabolism of cyclohexanecarboxylic acid by Acinetobacter anitratum. 1. The aerobic metabolism of cyclohexanecarboxylic acid by a bacterium isolated from garden soil (Acinetobacter anitratum) was investigated. 2. Evidence for the formation of cyclohex-1-ene-1-carboxylate, 2-hydroxycyclohexanecarboxylate and pimelate when either cell suspensions or cell-free extracts were incubated in the presence of cyclohexanecarboxylic acid is presented. 3. Crude cell-free extracts required ATP, CoA, FAD and Mg2+ as cofactors for the production of pimelate from cyclohexanecarboxylic acid, suggesting the existence of an activating reaction with formation of CoA esters, in this system.


A new method for the measurement of protein turnover. A new technique for the determination of rate constants of protein degradation is described. By using the method, half-lives of total soluble protein of Lemna minor during growth on full culture medium and distilled water were measured. The method involves incubating Lemna on a growth medium containing 3H2O. After a short exposure (20 min) to 3H-labelled culture medium, 3H was found in soluble amino acids, especially aspartate, glutamate, glutamine and alanine. After transfer to a 3H-free medium for 30 min, 80% of the 3H originally present in soluble amino acids was lost. These results suggest that 3H enters and leaves amino acids at the alpha-carbon atom, a conclusion supported by the observed labelling of glutamates. The exchange of H and 3H on the alpha-carbon atom is catalysed by transaminases and the speed of this exchange ensures that when the 3H2O is removed, the 3H in free amino acids is rapidly lost, thereby eliminating problems connected with metabolic pools and recycling. After an exposure of 20 min to 3H-labelled medium all protein amino acids, except for arginine, were found to be radioactive. The loss of radioactivity from protein amino acids was used to measure protein degradation.


The synthesis and secretion of cartilage procollagen. 1. Isolation of free and membrane-bound ribosomes from embryonic chick sternal-cartilage cells labelled for 4min with [14C]proline and their subsequent analysis for hydroxy[14C]proline indicated that cartilage procollagen biosynthesis occurs on bound ribosomes. 2. Nascent procollagen polypeptides on bound ribosomes isolated from cells labelled with [14C]lysine were found to contain hydroxy[14C]lysine indicating that hydroxylation of lysine commences while the growing chains are still attached to the ribosomes. 3. Analysis of bound ribosomes labelled with either [14C]proline or [14C]lysine on sucrose density gradients indicated that cartilage procollagen is synthesized on large polyribosomes in the range 250-400S. 4. Microsomal preparations isolated from cells pulse-labelled for 4 min with [14C]proline were used to determine the direction of release of nascent procollagen polypeptides. Puromycin induced the vectorial release of nascent procollagen polypeptides into the microsomal vesicles suggesting that the first step in the secretion of procollagen polypeptides is their transfer from the ribosomes through the membrane of the endoplasmic reticulum into the cisternal space. 5. The procollagen polypeptides secreted by cartilage cells were shown to be linked by inter-chain disulphide bonds. 6. Examination of the state of aggregation of pro-alpha chains in subcellular fractions isolated from cartilage cells labelled with [14C]proline for various periods of time have provided data on the timing and location of inter-chain disulphide-bond formation. This process commences in the rough endoplasmic reticulum after the release of completed pro-alpha chains from membrane-bound ribosomes. Pro-alpha chains isolated from fractions of smooth endoplasmic reticulum were virtually all present as disulphide-bonded aggregates, suggesting that either disulphide bonding is completed in this cellular compartment, or that procollagen needs to be in a disulphide-bonded form to be transferred to this region of the endoplasmic reticulum. 7. Comparison of these results with previously published data on disulphide bonding in tendon cells suggest that the rate of inter-chain disulphide-bond formation is significantly slower in cartilage cells.


The breakdown of lipid reserves in the endosperm of germinating castor beans. 1. Lipid extracts were obtained from castor-bean endosperm tissue at various times during germination and, after purification, the total lipid content was determined. Quantitative measurements of the triglyceride and phospholipid content together with the fatty acid composition were made. 2. The total lipid content of the endosperm rapidly decreased during germination; after 10 days less than 20% of the original weight of lipid remained. In contrast, the phospholipid content (initially less than 0.5% of the total lipid) increased slightly during this time. The fatty acid composition and the relative proportions of the triglyceride species of the total lipid extract remained constant during 10 days of germination. 3. Gibberellic acid (0.3 mM) markedly stimulated the rate of lipid breakdown but did not alter either the fatty acid composition or the relative proportion of triglyceride species. 4. The embryo had little effect on lipid metabolism in the endosperm tissue; only after 6 days of germination were differences observed in the rate of fat utilization in the presence and absence of the embryo.


Failure of adrenaline to induce hyperglycaemia after fructose injection in young mice. In control animals a 2-fold increase in liver phosphorylase activity 10min after adrenaline treatment was associated with a 55% increase in plasma glucose (P less than 0.001); at 20 min plasma glucose was 247% of the control value (P less than 0.001). Liver phosphorylase activity was decreased by 74%, 20 min after fructose injection (P less than 0.001), and, although phosphorylase activity increased 5-fold within 5 min of adrenaline injection, no increases in plasma glucose concentration over that found in fructose-injected animals which did not receive adrenaline occurred at either 5, 10 or 20 min. The data confirm inactivation of liver phosphorylase after fructose injection and suggest inhibition of the adrenaline-activated enzyme by the decrease in Pi and elevation of fructose 1-phosphate concentrations produced by the injection of fructose. These findings may be causally related to the hypoglycaemia and the lack of response to glucagon seen in patients with hereditary fructose intolerance after fructose ingestion.


8-Anilinonaphthalene-1-sulphonate interaction with whole and disrupted mitochondria: a re-evaluation of the use of double-reciprocal plots in the derivation of binding parameters for fluorescent probes binding to mitochondrial membranes. It is shown that 8-anilinonaphthalene-1-sulphonate is a permeant anion of whole mitochondrial membranes. It is also shown experimentally and algebraically that plots of reciprocal fluorescence against reciprocal membrane concentration, at a fixed 8-anilino-naphthalene-1-sulphonate concentration, are straight lines even when more than one binding site is involved.


Decreased synthesis of acetylcholine accompanying impaired oxidation of pyruvic acid in rat brain minces. The relation between pyruvate utilization and acetylcholine synthesis was investigated in minces of adult rat brain. The flux of pyruvate to acetylcholine was less than 1% of that to CO2; nevertheless, a number of agents which inhibited conversion of [1-14C]-pyruvate or [2-14C]pyruvate into 14CO2 were associated with corresponding decreases in the conversion of [2-14C]pyruvate into acetylcholine. The amount of acetylcholine produced by minces of whole rat brain, measured by g.l.c.-mass spectrometry, decreased similarly. Among the inhibitory compounds tested were 3-bromopyruvate, an irreversible inhibitor of pyruvate dehydrogenase; 2-oxobutyrate, a competitive inhibitor of pyruvate dehydrogenase; other 2-oxo acids; and amobarbital and pentobarbital. Linear-regression equations relating CO2 production to acetylcholine synthesis gave correlation coefficients of 0.89-0.93 for the combined observations. The inhibition of acetylcholine synthesis could not be attributed to inhibition of choline acetyltransferase. Incorporation of [2-14C]pyruvate into lipids, proteins and nucleic acids was effected less than that into acetylcholine. Under these experimental conditions, it was shown that pyruvate utilization can limit acetylcholine synthesis.


The effect of D-xylose, beta-D-xylosides and beta-D-galactosides on chondroitin sulphate biosynthesis in embryonic chicken cartilage. The incorporation of [3H]acetate into chondroitin sulphate was used as a measure of the rate of synthesis of this polysaccharide in whole tibias and femurs of embryonic chicken cartilage in vitro. The incorporation is inhibited by puromycin and by cycloheximide, but the inhibition is relieved by the addition of D-xylose, beta-D-xylosides and beta-D-galactosides to the incubation medium. Beta-D-Xylosides can stimulate the incorporation to 300% of that of controls incubated in the absence of cycloheximide or puromycin, D-Xylose, beta-D-xylosides and beta-D-galactosides appear to act as artificial initiators of chondroitin sulphate synthesis and enable polysaccharide-chain synthesis to be studied as an event separate from the synthesis of intact proteoglycan.


The regulation of the biosynthesis of ubiquinone in the rat. The urinary excretion of p-hydroxybenzoate was not altered by ubiquinone feeding, but, although decreased considerably, was not eliminated in protein deficiency. The incorporation of p-hydroxy[U-14C]benzaldehyde into ubiquinone in vivo increased in cold-exposed and p-chlorophenoxyisobutyrate (clofibrate)-fed rats, and these changes were parallel with the changes in the incorporation of [2-14C]mevalonate under these conditions. Starvation, cholesterol feeding and cholic acid feeding resulted in the decreased incorporation of p-hydroxy[U-14C]benzaldehyde into ubiquinone, confirming the decreased ubiquinone synthesis. Feeding exogenous ubiquinone increased the hepatic ubiquinone concentration, but did not cause any decrease in the incorporation of p-hydroxy[U-14C]benzaldehyde into ubiquinone, indicating the absence of a feedback control.


A mitochondrial monocarboxylate transporter in rat liver and heart and its possible function in cell control. Several hydroxy- and keto-substituted monocarboxylates were found to undergo co- as well as counter-exchange across the mitochondrial membrane. The results argue against a simple Donnan system and may be explained by the existence of a transporter for monocarboxylates. In support of this explanation it was apparently possible to 'pump' pyruvate to the sucrose-inaccessible space by using the dicarboxylate transporter. Further, several aromatic and aliphatic analogues of pyruvate, but not of di- or tri-carboxylate transport inhibitors, have been shown to prevent pyruvate-exchange reactions. Palmitoylcarnitine was found to have a much stronger affinity for the carrier than either carnitine or pyruvate and the possible consequences of this for carnitine-palmitoylcarnitine exchange and on the control of the pyruvate dehydrogenase complex are explored. In view of the range of transport inhibitors and substrates it is suggested that the carrier has a fairly broad specificity. 'Inhibitor-stop' kinetic studies show that the speed of translocation of pyruvate at 1 degrees C is of the same order as malate. The possible correlation between the role of a hydroxy-keto acid transporter in substrate exchange and some whole animal experiments is briefly discussed. It is proposed that for reasons of control the cell will require membrane monocarboxylate transporters no less than di- or tri-carboxylate carriers.


Deoxyribonucleic acid-dependent ribonucleic acid polymerase activity in rat liver after protein restriction. Rats were fed for 6 days on a diet containing either 3 or 20% high-quality protein. Nuclei were isolated from liver and DNA-dependent RNA polymerases (EC 2.7.7.6) extracted with 1 M-(NH4)2SO4. The proteins were then precipitated with 3.5 M-(NH4)2SO4 and after dialysis applied to a DEAE-Sephadex column. The column was developed with a gradient of (NH4)2SO4. Polymerase I separated well from alpha-amanitin-sensitive polymerase II. The enzyme activities were compared between the two dietary groups. Rats that had received 3% protein showed a lower polymerase I activity per g wet wt. of liver, per mg of DNA and per mg of protein. Polymerase II was lower in activity per g wet wt. of liver and per mg of DNA, but was higher per mg of protein. Polyacrylamide-gel electrophoretograms showed a higher proportion of contaminating proteins in polymerase II fractions isolated from 20%-protein-fed rats. The data explain the lower activity obtained per mg of protein in these rats. It is concluded that a decrease in dietary protein content from 20 to 3% induces a fall in content and specific activity of RNA polymerase I and II in liver.


Alkaline ribonuclease and ribonuclease inhibitor in mammary gland during the lactation cycle and in the R3230AC mammary tumour. Alkaline RNAase (ribonuclease) and RNAase inhibitor were assayed to determine the potential role of the degradative process in regulating the amount of RNA in the mammary gland and mammary tumour. Very little free alkaline RNAase activity was found in the cytosol fraction of the mammary gland of virgin, pregnant, lactating or involuting Fischer rats. However, addition of p-chloromercuribenzoate to the assay medium revealed latent RNAase which, when expressed on a DNA basis, decreased during pregnancy and lactation. The cytosol latent RNAase is stable in 0.125 M-H2SO4. The non-cytosol RNAase activity also decreased during pregnancy and lactation. Addition of Triton X-100 produced slightly higher activity at all stages tested. The inhibitor activity in rat mammary gland was very low before pregnancy, increased gradually during pregnancy and more dramatically at parturition, continued to increase throughout lactation and returned to resting-gland values by the sixth day of involution. The increase during pregnancy may be due to the increased cellularity of the gland, whereas the gain during lactation was more than could be accounted for by increases in cell number. The R3230AC transplantable mammary tumour resembles the normal gland in early lactation with respect to both its cytosol and non-cytosol alkaline RNAase activities and its moderately high content of RNAase inhibitor. The relatively high inhibitor and low RNAase activities in both the gland of the lactating rat and in the tumour are of potential significance in maintaining high amounts of RNA and increased rates of protein synthesis in these tissues.


The mitochondrial pyruvate carrier. Kinetics and specificity for substrates and inhibitors. 1. Studies on the kinetics of pyruvate transport into mitochondria by an 'inhibitor-stop' technique were hampered by the decarboxylation of pyruvate by mitochondria even in the presence of rotenone. Decarboxylation was minimal at 6 degrees C. At this temperature the Km for pyruvate was 0.15 mM and Vmax. was 0.54nmol/min per mg of protein; alpha-cyano-4-hydroxycinnamate was found to be a non-competitive inhibitor, Ki 6.3 muM, and phenyl-pyruvate a competitive inhibitor, Ki 1.8 mM. 2. At 100 muM concentration, alpha-cyano-4-hydroxycinnamate rapidly and almost totally inhibited O2 uptake by rat heart mitochondria oxidizing pyruvate. Inhibition could be detected at concentrations of inhibitor as low as 1 muM although inhibition took time to develop at this concentration. Inhibition could be reversed by diluting out the inhibitor. 3. Various analogues of alpha-cyano-4-hydroxycinnamate were tested on rat liver and heart mitochondria. The important structural features appeared to be the alpha-cyanopropenoate group and the hydrophobic aromatic side chain. Alpha-Cyanocinnamate, alpha-cyano-5-phenyl-2,4-pentadienoate and compound UK 5099 [alpha-cyano-beta-(2-phenylindol-3-yl)acrylate] were all more powerful inhibitors than alpha-cyano-4-hydroxycinnamate showing 50% inhibition of pyruvate-dependent O2 consumption by rat heart mitochondria at concentrations of 200, 200 and 50 nM respectively. 4. The specificity of the carrier for its substrate was studied by both influx and efflux experiments. Oxamate, 2-oxobutyrate, phenylpyruvate, 2-oxo-4-methyl-pentanoate, chloroacetate, dichloroacetate, difluoroacetate, 2-chloropropionate, 3-chloropropionate and 2,2-dichloropropionate all exchanged with pyruvate, whereas acetate, lactate and trichloroacetate did not. 5. Pyruvate entry into the mitochondria was shown to be accompanied by the transport of a proton (or by exchange with an OH-ion). This proton flux was inhibited by alpha-cyano-4-hydroxycinnamate and allowed measurements of pyruvate transport at higher temperatures to be made. The activation energy of mitochondrial pyruvate transport was found to be 113 kJ (27 kcal)/mol and by extrapolation the rate of transport of pyruvate at 37 degrees C to be 42 nmol/min per mg of protein. The possibility that pyruvate transport into mitochondria may be rate limiting and involved in the regulation of gluconegenesis is discussed. 6. The transport of various monocarboxylic acids into mitochondria was studied by monitoring proton influx. The transport of dichloroacetate, difluoroacetate and oxamate appeared to be largely dependent on the pyruvate carrier and could be inhibited by pyruvate-transport inhibitors. However, many other halogenated and 2-oxo acids which could exchange with pyruvate on the carrier entered freely even in the presence of inhibitor.


The isolation and properties of granulocytic colony-stimulating activities from medium conditioned by human peripheral leucocytes. The colony-stimulating activity detected by its ability to promote colony formation by human granulopoietic progenitor cells was partially purified from medium conditioned by human peripheral leucocytes. The purification procedure utilized (NH4)2SO4 precipitation, DEAE-cellulose chromatography, hydroxyapatite chromatography and gel filtration on Sephadex G-150 and yielded a purification of about 1000-fold. The medium from cultures of non-leukaemic cells contained three molecular species of colony-stimulating activity with approximate molecular weights of 93000, 36500 and 14700. On the basis of their sensitivity to enzymes, these species of activity appeared to be proteins. In contrast, medium from cultures of leukaemic cells contained only one detectable molecular species with colony-stimulating activity, usually with an approximate molecular weight of 36500. The results are discussed in relation to concurrent studies on the association of the different species of colony-stimulating activity with the cell surface membrane.


The metabolism of labelled hexadecyl sulphate salts in the rat, dog and human. The metabolic fate of [1-14-C]hexadecylsulphate and hexadecyl[35-S]sulphate, administered intravenously as the sodium and trimethylammonium salt to dogs and orally as the erythromycin salt to dogs, rats and humans, was studied. Studies with rats indicated that the compounds were well absorbed and rapidly excreted in the urine. However, after oral administration of the 14-C-and 35-S-labelled hexadecyl sulphate erythromycin salt to dogs, considerable amounts of radioactivity were excreted in the faeces as unmetabolized hexadecyl sulphate. Studies with two humans showed that orally administered erythromycin salt of [1-14C]hexadecyl sulphate was well absorbed in one person but poorly absorbed in the other. Radioactive metabolites in urine were separated by t.l.c. in two solvent systems. The main metabolite of hexadecyl sulphate in the dog, rat and human was identified as the sulphate ester of 4-hydroxybutyric acid. In addition, psi-[14-C]butyrolactone as a minor metabolic product of [1-14-C]hexadecyl sulphate was also isolated from the urine of rat, dog and man. However, there was still another metabolite in dog urine, which comprised about 20% of the total urinary radioactivity and carried both 14-C and 35-S labels. This metabolite was absent from rat urine. The metabolite in dog urine was isolated and subsequently identified by t.l.c. and g.l.c. and by isotope-dilution experiments as the sulphate ester of glycollic acid. Small amounts (about 5% of the total recovered radioactivity in excreta) of labelled glycollic acid sulphate were also found in human urine after ingestion of erythromycin [1-14-C]hexadecyl sulphate.


Thymidine transport in phytohaemagglutinin-stimulated pig lymphocytes. Thymidine and uridine transporters in peripheral pig lymphocytes have structural features in common, but are not identical. Accelerated entry of [3H]thymidine begins 12h after the addition of phytohaemagglutinin. The increased thymidine uptake into the cells is characterized by an increase in Vmax. Without alteration of the apparent Km(0.6+/-0.08muM). Thymidine kinase activity is increased 12h after stimulation. Both the increased thymidine uptake and the increased thymidine kinase activity are inhibited in cultures incubated with puromycin: rates of degradation of the two systems are unchanged after phytohaemagglutinin addition, and indicate similar half-lives of about 2h. Thymidine kinase is rate-limiting for thymidine entry up to 18h after phytohaemagglutinin addition; increase in its synthesis is detectable about 6h before net incorporation of thymidine into DNA is significantly promoted.


A model system for investigating the biliary excretion of an anion in the rat. 1. The biliary excretion of phenolphthalein di[35S]sulphate was studied in rats. 2. The conjugate was administered by continuous infusion at rates of 3, 4.5, 6, 9 and 12 mug/min, and kinetic analysis of the rate of biliary excretion was consistent with a two-compartment open-model system. 3. The results obtained after single injections of the ester were also consistent with the model. 4. An essential feature of the model is the presence of a compartment into which the ester may pass as an alternative to direct excretion via the bile. 5. It is suggested that such a compartment may be located within the liver.


Regulation of enzyme turnover during tissue differention. Studies on the effects of hormones on the turnover of fatty acid synthetase in rabbit mammary gland in organ culture. 1. Explants of mammary gland from mid-pregnant rabbits were cultured with insulin, prolactin and cortisol. 2. Antibodies raised to fatty acid synthetase were used to measure the amount as well as the rate of synthesis and the rate of degradation of the enzyme in the explants over defined periods in organ culture. These measurements were also made after the hormones had been removed from the culture medium. The changes which occur in the activity of fatty acid synthetase are due to changes in the amount of the enzyme present. They are not due to activation or inactivation of the enzyme. 3. The rate of lipogenesis (measured from [1-14C]acetate) in the explants during culture varies independently of the amount of fatty acid synthetase both in the presence and after removal of the hormones. Hence the amount of fatty acid synthetase does not limit lipogenesis. The proportion of medium-chain fatty acids C8:0 and C10:0 (which are characteristic of rabbit milk) synthesized by the explants in the presence of hormones increases at about the same rate as the amount of fatty acid synthetase present. However, when hormones are removed from the medium the proportion of these acids synthesized declines as rapidly as the rate of lipogenesis and not as the amount of fatty acid synthetase presen. 4. The rates of synthesis of fatty acid synthetase and of the total particulate-free supernatant protein in the explants were compared by measuring the incorporation of L-[U-14C]leucine into the protein of the explants. These rates increase by 5-fold and 3.6-fold respectively when explants are cultured with hormones, and they then reach approximately constant rates. When the hormones are removed there is a rapid fall in the rate of synthesis of fatty acid synthetase and of the total particulate-free supernatant protein to values which are similar to those obtained with freshly prepared explanted tissue. 5. In unstimulated explants fatty acid synthetase appears to be degraded with a half-life of 15-21h. During the hormonally stimulated differentiation of the tissue the rate of degradation of the enzyme is considerably decreased or is switched off completely. After the amount of fatty acid synthetase has increased to a maximum the enzyme complex is again degraded with a half-life of 23-29h. The removal of hormones after the explants have been hormonally stimulated for different times results in an increase in the rate of degradation of fatty acid synthetase. However, this increase only occurs if degradation was previously proceeding at a considerably decreased rate. The degradation of the total particulate-free supernatant protein continues throughout the period of differentiation of the explant tissue in culture. It appears to be somewhat decreased during the period of rapid maturation of the tissue during culture.


The localization of glycollate-pathway enzymes in Euglena. Isolation of organelles from broken-cell suspensions of phototrophically grown Euglena gracilis Klebs was achieved by isopycnic centrifugation on sucrose gradients. 2. Equilibrium densities of 1.23g/cm3 for peroxisome-like particles, 1.22g/cm3 for mitochondria and 1.17g/cm3 for chloroplasts were recorded. 3. The enzymes glycollate dehydrogenase, glutamate-glyoxylate aminotransferase, serineglyoxylate aminotransferase, aspartate-alpha-oxoglutarate aminotransferase, hydroxy pyruvate reductase and malate dehydrogenase were present in peroxisome-like particles. 4. Unlike higher plants glycollate dehydrogenase and glutamate-glyoxylate aminotransferase were present in the mitochondria of Euglena. 5. Rates of glycollate and  D-lactate oxidation were additive in the mitochondria, and, although glycollate dehydrogenase was inhibited by cyanide, D-lactate dehydrogenase activity was unaffected. 6. Glycollate oxidation was linked to O2 uptake in mitochondria but not in peroxisome-like particles. This glycollate-dependent O2 uptake was inhibited by antimycin A or cyanide. 7. The physiological significance of glycollate metabolism in Euglena mitochondria is discussed, with special reference to its role in photorespiration in algae.


Accelerated uptake of 5-hydroxytryptamine by human blood platelets enriched in a sialic acid. The enzymically catalysed incorporation of N-acetylneuraminic acid into human platelets, whether suspended in their own citrated plasma or in buffered saline containing 0.17 mM-sucrose, accelerated the uptake of 5-hydroxytryptamine. This acceleration decreased with time. The observations may be explained by assuming that N-acetylneuraminic acid is a component of a transport receptor for 5-hydroxytryptamine.


Glucose turnover rate in newborn rats.

Glucose turnover rate in newborn rats. In starved newborn rats an increase in glucose turnover rate was observed 4 and 6h after birth, but a dramatic fall occurred at 16h. In suckling newborn rats, no decrease in glucose turnover rate was observed at 16h. The metabolic clearance of glucose did not change in fed or starved animals. The results are discussed in relation to metabolic adaptation to extra-uterine life.


Aspirin-induced hepatotoxicity in juvenile rheumatoid arthritis. A prospective study. Recent reports of and our own experience with biochemical alterations of liver function secondary to salicylate therapy stimulated this prospective study. Thirty-four children with juvenile rheumatoid arthritis, 6 children with acute cartilagenous necrosis of the hipfollowing slipped capital femoral epiphysis, and 2 children with ulcerative colitis and hip disease who were on salicylates were followed over a period of 1-27 months with serial determinations of serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), lactic dehydrogenase (LDH), alkaline phosphatase (AP), bilirubin, and serum salicylate. Prothrombin time was measured in 14 children. Twenty-two of 34 children with rheumatoid arthritis and none of the 8 controls demonstrated abnormalities of various liver functions at serum salicylate levels between 7.0 and mg%. Three children demonstrated severe abnormalities characterized by marked elevation of SGOT, SGPT, LDH, and AP, prolongation of prothrombin time, and epistaxis. This type of reaction occurred within 5-14 days of initiation of aspirin therapy and occurred at serum salicylate levels between 18 and 43 mg%. Moderate changes in various liver function tests were observed in 19 other children. None of those children who were tested showed prolongation of prothrombin time. The serum salicylate level in this group varied between 7.0 and 38.2 mg%. The abnormal liver function tests returned to normal in 6 children upon withdrawal of aspirin and in 12 others even when salicylates were continued. Therefore, despite the occurrence of biochemical abnormalities following chronic salicylate therapy, it does not appear to be necessary to discontinue their use except in those children who develop bleeding.


Thiemann's disease. A brief reminder.

Thiemann's disease. A brief reminder. Two brothers with Thiemann's disease are described and the accumulated world literature is briefly reviewed. Thiemann's disease is considered to be a form of idiopathic avascular necrosis of the PIP joints of the hands, with occasional involvement of other joints. The characteristic deformity and x-ray findings are reviewed. Thiemann's disease is a genetic disorder and, in families in which detail is available, is inherited as an autosomal dominant with virtual complete penetrance.


Aseptic meningitis in systemic lupus erythematosus. Report of three cases. Three patients with aseptic meningitis were subsequently diagnosed as having lupus erythematosus. One patient had a single meningitic episode, another had chronic meningitis, and the third two acute episodes 5 years apart. All 3 patients developed further neurophychiatric manifestations of SLE, leading to death in 1. Aseptic meningitis appears to be an early manifestation of SLE and may herald more serious brain damage. No new cases of aseptic meningitis occurred in this series after initiation of therapy for SLE. In contrast, bacterial meningitis did occur as a late complication of the disease.


Osteoarthritis of the trapezioscaphoid joint. Isolated osteoarthritis (OA) of the trapezioscaphoid (TS) joint is little recognized. Nine cases that were examined clinically and radiologically are described. Of these 4 had isolated TS OA, 3 also had generalized OA, and 2 also had rheumatoid arthritis. Symptoms and signs, including pain in the wrist and thumb base, radial and volar swelling, and tenderness over the scaphoid, allowed distinction from other causes of pain at the thumb base. A wrist-working splint was beneficial in relieving pain.


[Mesenterico-caval shunt with interposition of a prosthesis for intractable ascites caused by cirrhosis]. The interposition mesocaval shunt is followed both by a splancnic and sinusoidal decompression, being for this reason considered not only the most effective peripheral porto-systemic shunt, but also one of the indicated operations in the treatment of intractable cyrrhotic ascites. Intractable cyrrhotic ascites was the main indication in 9 cases with very different clinical pictures treated by the Author with mesocaval shunt, having observed no operative mortality and resulting a complete disappearance of ascites in 8 out of 9 cases and of the cohexistent idrothorax in one case. Angiographic pictures showed the patency of the shunt with constant demonstration, after few months, of a reversal portal blood flow (sometimes present before operation). This shunting procedure proved to be a very well tolerated operation with only a transient postoperative modification of liver function tests, excepted for one case in which the patient was lost after seven months because of hepatic encephalopaty with progressive liver failure.


[Experimental revascularization of acute myocardial infarct]. Late clinical and istological changes. The Authors studies clinical and istological changes in dogs underwent to actue myocardial ischemia and follow revascularization after six hours. The experience infer that while clinical and ECGraphic changes had a patency grow worsing after revascularization, istological changes presents a reduction of necrotic area with functional improvement of little myocardial areas strictly interested by ischemia.


[Changes in the specific activity and content of free nucleotides and polyamines of the infarct and pre-infarct area of the revascularized dog heart]. Changes on specific radioactivity and levels of free nucleotides and polyamines in infarcted and borderline tissue of reperfused dog heart. The changes on specific radioactivity and levels of free nucleotides and polyamines, spermine and spermidine, of reperfused heart show a different behaviour of the anoxic myocardium. An increase of both specific activity and levels of free nucleotides and polyamines after 30 minutes of ischemia is observed. A longer period of anoxia (6 hours) causes a decreased synthesis and concentration of free nucleotides and polyamines. A remarkable recovery of specific activity of these compounds after reperfusion is noted. The borderline tissue shows a similar behaviour but with smaller changes. Therefore, in our experimental conditions, ischemia does not cause an irreversible alteration in protein synthesis mechanism of myocardial cells. In addition, the reperfusion may cause a recovery of biochemical mechanisms that control the functional capacity of the cell. The polyamine changes may postulate a central role of these amines in both anoxic and reperfused heart.


[Critical considerations on the behavior of immunoglobulins in rheumatoid arthritis]. Critical evaluation of immunoglobulins in Rheumatoid Arthritis. It has been studied the behavior of the serum and synovial immunoglobulins of 62 patients afftected arthritis reheumatoid in various clinic stages; the valued have been expressed in mg% and mg/g of proteins. The serum levels of the IgG and IgA are higher in the cases of R.A. than in normals. The synovial concentrations of the IgA and IgM are lower than the respective serum values, while the phenomenon is less evident for the IgG probably because the last are synthetised in the synovial means. It exists, however, a interdipendence between the concentration of the serum and synovial immunoglobulins. The concentrations of the immunoglobulines, both serum and synovial, are not in relation with the most usual humoral phlogosis-tests, as well as they are not correlable with the values of the naturals antiproteases and with some fractions of the complement (both of the classic and alternative pathway). The variations of the immunoglobulins are, in the R.A., aspecific and of different intensity therefore their determination is not proposable neither as test of measure of the phlogosis nor as index of disease.


[Anatomo pathological and medicolegal data on 4 cases of unwitnessed death of infants]. The Author gives his contribution to the study of "untestified" death of children still unweaned and describes four cases. Except for one case (porpora encefalica) the macroscopic appearance is insignificant. On the other hand, the histologic findings demonstrate presence of several foci of infiltration of the heart, lung, liver and brain. The Author suggests the diagnosis of viral disease even-thoug more accurate bacterial and viral exames are indispensable for a precise definition of the cases reported.


[Changes in the EEG in the course of therapy with lithium salts]. The effect of lithium during chronic treatment on the EEG. has been studied in 21 subbjects affected by manic-depressive psychosis. The relation between lithiemic values and EEG. modification has been discussed from varius points of view, considering records of what has been observed in the present research and what reported in literature. The mean hematic lithium level has been considered insufficient to obtein the essential informations. The determination of the intracellular lithium concentration may be a more informative one.


The protective action of essential oils of onion and garlic in cholesterol-fed rabbits. The effects of the essential oils of onion and garlic have been investigated in cholesterol-fed rabbits and have been compared with the effects of clofibrate. The marked rise in serum cholesterol and blood coagulability that followed 3 months of cholesterol feeding (0.2 g/kg/day) was significantly reduced by the essential oils of both onion and garlic. Fibrinolytic activity was actually increased even above the normal control levels. The essential oils of onion and garlic (equivalent to 1 g/kg/day of raw bulbs) proved more effective than clofibrate in the usual clinical dose of 33 mg/kg/day. Garlic was even more effective than onion. Cholesterol feeding increased the average lipid content of aorta from 5.95 to 13.75 mg/100 mg dry weight. With addition of clofibrate, onion or garlic the values were 7.79, 6.23 and 5.28 mg/100 ml, respectively. Thus it can be seen that the essential oils effectively prevent lipid acculation in the rabbit oarta.


Effect of fasting, phenobarbital, or hydrocortisone on serum creatine-phosphokinase and aldolase activity in myopathic Syrian hamsters. The hereditary polymyopathy and cardiomyopathy of inbred Syrian hamsters (UM X 7.1) are associated with a marked elevation of serum creatine phosphokinase (CPK) and aldolase levels. Fasting (overnight) further increases (+100-500%) the serum concentration of these enzymes in myopathic hamsters; however, no such effect is demonstrable in normal hamsters, or in first generation offspring produced by crossbreeding the two strains (myopathic and normal). The changes are reversible, and the enzyme values return to previous levels within 72 hr. Neither phenobarbital nor hydrocortisone prevents the rises, as shown by CPK determinations; on the contrary, hydrocortisone elicits even greater serum enzyme increases.


[Dupuytren's disease and cervical arthrosis]. The Authors, after having briefly discribed Dupuytren's disease in its anatomical and pathological manifestations next discribe the various theories as to causes of the disease. In conclusion they are of the opinion that the theory of arthritis is the most probable, statistically speaking.


Plasma concentrations of lysolecithin and other phospholipids in the healthy population and in men suffering from atherosclerotic diseases. The concentrations of individual phospholipids and of cholesterol have been determined in plasma samples taken from 77 apparently healthy individuals, and from 76 male patients presenting with atherosclerotic diseases. Significant differences in the relative and absolute concentrations of lysolecithin were found between different populations. In healthy individuals the plasma levels of lysolecithin were lower in women than in men and lower in the younger age groups studied. The relative and absolute concentrations of plasma lysolecithin were lower in men suffering from chronic ischaemic heart disease and peripheral arterial disease when compared with age-matched healthy male subjects. The lowest levels of plasma lysolecthin were, however, associated with patients suffering from acute myocardial infarction or acute myocardial ischaemia studied within 48 hr of the onset of chest pain. In a further study, significantly decreased relative concentrations of lysolecithin were found in blood platelets and erythrocytes as well as in plasma of patients suffering from chronic ischaemic heart disease. The results are discussed in terms of a possible thrombo-protective role for plasma lysolecithin in man.